Synthetic DNA encoding an orange seapen-derived green fluorescent protein with codon preference of mammalian expression systems and biosensors

ABSTRACT

Synthetic versions of a full length and termini truncated humanized green fluorescent protein based on  Ptilosarcus gurneyi  are disclosed which have been modified to the favored or most favored codons for mammalian expression systems. The disclosed encoded protein has 239 amino acid residues compared with the wild type  Ptilosarcus gurneyi  which has 238 amino acids. In the present invention, a valine residue has been added at the second position from the amino terminus and codon preference bias has been changed in a majority of the wild type codons of  Ptilosarcus gurneyi  fluorescent protein. The humanized  Ptilosarcus gurneyi  green fluorescent protein is useful as a fluorescent tag for monitoring the activities of its fusion partners using imaging based approaches.

CROSS REFERENCE TO RELATED APPLICATIONS

This utility patent application is a divisional patent application ofU.S. patent application Ser. No. 09/977,897, filed Oct. 15, 2001 nowU.S. Pat. No. 6,780,974, which claims the benefit of prior U.S.Provisional Patent Application Ser. No. 60/297,645, filed Jun. 12, 2001,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an isolated and purified DNA encoding ahumanized bioluminescent green fluorescent protein (hPtFP) derived fromthe orange seapen Ptilosarcus gurneyi in which all the codons are thefavored or most favored codons for mammalian expression systems.Truncation mutants of the humanized Ptilosarcus gurneyi fluorescentprotein (hPtFP) of the present invention are functional as fluorescentreporter molecules in a biosensor system. The green fluorescent proteinof the present invention is useful as an improved fusion partner incellular proteins allowing direct observation of the behavior of thetagged protein.

2. Description of the Background Art

A major component of the new drug discovery paradigm is a continuallygrowing family of fluorescent and luminescent reagents that are used tomeasure the temporal and spatial distribution, content, and activity ofintracellular ions, metabolites, macromolecules and organelles. Classesof these reagents include labeling reagents that measure thedistribution and amount of molecules in living or fixed cells,environmental indicators to report signal transduction events in timeand space, and fluorescent protein biosensors to measure targetmolecular activities within living cells. A multiparameter approach thatcombines several reagents in a single cell is a powerful new tool fordrug discovery.

Those skilled in this art will recognize a wide variety of fluorescentreporter molecules that can be used in the field of drug discovery.Particularly, herein are disclosed novel humanized fluorescent proteins.Similarly, fluorescent reagents specifically synthesized with particularchemical properties of binding or association have been used asfluorescent reporter molecules. (Barak et al., (1997), J. Biol. Chem.272:27497–27500; Southwick et al., (1990), Cytometry 11:418–430; Tsien(1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp.127–156). Fluorescently labeled antibodies are particularly usefulreporter molecules due to their high degree of specificity for attachingto a single molecular target in a mixture of molecules as complex as acell or tissue. However, fluorescently labeled antibodies presentseveral limitations.

It is known that luminescent probes can be synthesized within the livingcell or can be transported into the cell via several non-mechanicalmodes including diffusion, facilitated or active transport,signal-sequence-mediated transport, and endocytic or pinocytic uptake.Mechanical bulk loading methods, which are well known in the art, canalso be used to load luminescent probes into living cells. (Barber etal. (1996), Neuroscience letters 207:17–20; Bright et al. (1996),Cytometry 24:226–233; McNeil (1989) in Methods in Cell Biology, Vol. 29,Taylor and Wang (eds.) pp. 153–173). These methods includeelectroporation and other mechanical methods such as scrape-loading,bead-loading, impact loading, syringe-loading, hypertonic and hypotonicloading. Additionally, cells can be genetically engineered to expressreporter molecules such as Green Fluorescent Protein, coupled to aprotein of interest as previously described (Chalfie and Prasher U.S.Pat. No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science,20:448–455).

Luminescence is the process whereby a molecule is electronically excitedand releases light when it returns to a lower energy state.Bioluminescence is the process by which living organisms emit light thatis visible to other organisms. In bioluminescence the excited state iscreated by an enzyme-catalyzed reaction. The color of the emitted lightin a bioluminescent reaction is characteristic of the excited molecule,and is independent from its source of excitation and temperature.

Molecular oxygen is known to be essential in some well characterizedbioluminescent systems, such as the bioluminescence of luciferase.Luciferases are oxygenases, that act on a substrate, luciferin, in thepresence of molecular oxygen and transform the substrate to an excitedstate. Upon return to a lower energy level, energy is released in theform of light. Ward et al., Chapter 7 in Chemi- and Bio-luminescence,Burr ed. Marcel Dekker, Inc. NY, pp. 321–358; Hastings, J. W. (1995)Cell Physiology: Source Book, N. Sperelakis (ed.), Academic Press, pp.665–681; Luminescence, Narcosis and Life in the Deep Sea, JohnsonVantage Press, NY, pp. 50–56. Bioluminescent species span many generaand include microscopic organisms, including bacteria, primarily marinebacteria such as Vibrio species, fungi, algae, and dinoflagellates, tomarine organisms including arthropods, mollusks, echinoderms, andchordates, and terrestrial organisms including annelids and insects.

Luminescence (bioluminescence, chemiluminescence, and fluorescence) isused for qualitative and quantitative determination of specificsubstances and processes in biology and medicine. For example, variousluciferase genes from various organisms have been cloned and exploitedas reporters in numerous assays. On the other hand, treating cells withdyes and fluorescent biomolecules allowing imaging of the cells, andgenetic engineering of cells to produce fluorescent proteins as reportermolecules are useful detection methods known by those persons skilled inthe art. For instance, treating cells with dyes and fluorescentbiomolecules allowing imaging the cells, and genetic engineering ofcells to produce fluorescent proteins as reporter molecules are usefuldetection methods known in the art. Wang et al., Methods in CellBiology, New York, Alan R. Liss, 29:1–12, 1989. One such fluorescentreporter protein is the green fluorescent protein (GFP) of the jellyfishAequorea victoria which absorbs blue light with an excitation maximum at395 nm, with a minor peak at 470 nm, and emits green fluorescence withan emission maximum at 510 nm, with a minor peak near 540 nm and doesnot require an exogenous factor. However, the absorption and emissionspectra for Aequorea GFP present certain limitations. The excitation andemission maxima of the wild type Aequorea GFP are not within the optimalrange of wavelengths of standard fluorescence optics.

The green fluorescent proteins (GFP) constitute a class ofchromoproteins found among certain bioluminescent coelenterates. Theseproteins are fluorescent and function as the ultimate bioluminescenceemitter in these organisms by accepting energy from enzyme-bound,excited state oxyluciferin. Ward et al., (1982) Biochemistry21:4535–4540.

Uses of Aequora GFP for the study of gene expression and proteinlocalization are discussed in Chalfie et al., Science 263:802–805, 1994.Some properties of wild-type Aequora GFP are disclosed by Morise et al.,Biochemistry 13:2656–2662, 1974, and Ward et al., Photochem. Photobiol.31:611–615, 1980. An article by Rizzuto et al., Nature 358:325–327,1992, discusses the use of wild-type Aequora GFP as a tool forvisualizing subcellular organelles in cells. Kaether and Gerdes, FEBSLetters 369:267–271, 1995, report the visualization of protein transportalong the secretory pathway using wild-type Aequora GFP. The expressionof Aequora GFP in plant cells is discussed by Hu and Cheng, FEBS Letters369:331–334, 1995, while Aequora GFP expression in Drosophila embryos isdescribed by Davis et al., Dev. Biology 170:726–729, 1995.

U.S. Pat. No. 5,491,084 discloses expression of GFP from Aequoreavictoria in cells for use as a reporter molecule fused to anotherprotein of interest. PCT/DK96/00052 relates to methods of detectingbiologically active substances affecting intracellular processes byutilizing a GFP construct having a protein kinase activation site. GFPproteins are used in various biological systems. For example,PCT/US95/10165 describes a system for isolating cells of interestutilizing the expression of a GFP-like protein. PCT/GB96/00481 describesthe expression of GFP in plants. PCT/US95/01425 describes modified GFPprotein expressed in transformed organisms to detect mutagenesis.Mutants of GFP have been prepared and used in several biologicalsystems. (Hasselhoff et al., Proc. Natl. Acad. Sci. 94:2122–2127, 1997;Brejc et al., Proc. Natl. Acad Sci. 94:2306–2311, 1997; Cheng et al.,Nature Biotech. 14:606–609, 1996; Heim and Tsien, Curr. Biol. 6:178–192,1996; Ehrig et al., FEBS Letters 367:163–166, 1995). Methods describingassays and compositions for detecting and evaluating the intracellulartransduction of an extracellular signal using recombinant cells thatexpress cell surface receptors and contain reporter gene constructs thatinclude transcriptional regulatory elements that are responsive to theactivity of cell surface receptors are disclosed in U.S. Pat. No.5,436,128 and U.S. Pat. No. 5,401,629.

Certain types of cells within an organism may contain components thatcan be specifically labeled that may not occur in other cell types. Forexample, epithelial cells often contain polarized membrane components.That is, these cells asymmetrically distribute macromolecules alongtheir plasma membrane. Connective or supporting tissue cells oftencontain granules in which are trapped molecules specific to that celltype (e.g. heparin, histamine, serotonin, etc.). Skeletal muscle cellscontain a sarcoplasmic reticulum, a specialized organelle whose functionis to regulate the concentration of calcium ions within the cellcytoplasm. Many nervous tissue cells contain secretory granules andvesicles in which are trapped neurohormones or neurotransmitters.Therefore, fluorescent molecules can be designed to label not onlyspecific components within specific cells, but also specific cellswithin a population of mixed cell types.

Those skilled in the art will recognize a wide variety of ways tomeasure fluorescence. For example, some fluorescent reporter moleculesexhibit a change in excitation or emission spectra, some exhibitresonance energy transfer where one fluorescent reporter losesfluorescence, while a second gains in fluorescence, some exhibit a loss(quenching) or appearance of fluorescence, while some report rotationalmovements. (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol.Structure 24:405–434; Giuliano et al. (1995), Methods in Neuroscience27:1–16). The GFPs exhibit absorption at a particular wavelength, andemission at a different wavelength characteristic for each greenfluorescent protein which sometimes allows for the pairing of GFP's withtwo distinct signals being detectable.

In addition to the limitations in detection with standard fluorescenceoptics presented by the absorption-emission wavelength spectrum ofAequora GFP, another difficulty is the potentially low level offluorescent signal emitted by GFP transfected into a heterologous celltype. This is the result of low level expression normally associatedwith the expression of a non-native species protein being expressed by acell, in this case a jellyfish protein being expressed in higher levelorganisms such as mammals. This is partly due to different codon usagein the native marine organism sequences that are different from the hostor transfected cell's codon usage. In spite of this background art,there remains a very real and substantial need for a fluorescentreporter molecule having a narrower absorption-emission wavelengthspectrum and having an optimized expression in a host or transfectedcell resulting in fluorescent signals that are easily detected withstandard fluorescence optics.

U.S. Pat. No. 5,786,464 (Seed et al.) and U.S. Pat. No. 5,795,737 (Seedet al.) dislcose replacing non-preferred codons with preferred codons toincrease expression in mammalian cell lines of other proteins, such asthe green fluorescent protein of the jellyfish Aequorea victoria.

U.S. Pat. No. 5,874,304 (Zolotukhin et al.) discloses a humanized greenfluorescent protein gene adapted from the jellyfish Aequorea victoria.U.S. Pat. No. 5,968,750 (Zolotukhin et al.) discloses a method oflabeling a mammalian cell comprising expressing a humanized greenfluorescent protein gene in the cell wherein the genes have an increasednumber of GCC or GCT alanine-encoding codons in comparison to the wildtype jellyfish gene sequence.

U.S. Pat. No. 6,232,107 (Bryan et al.) discloses isolated and purifiednucleic acids encoding green fluorescent proteins from the genus Renillaand Ptilosarcus and the green fluorescent proteins encoded thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows comparative fluorescence of COS1 cells expressing thesynthetic DNA versus a commercially available nuclear dye (Hoechst 33342stain).

FIG. 2 shows in situ fluorescence of the humanized Ptilosarcus gurneyifluorescent protein in COS1 cells.

FIG. 3 is a histogram of the fluorescent intensity in COS1 cellstransiently transfected with wild type Ptilosarcus gurneyi greenfluorescent protein DNA or the synthetic green fluorescent protein DNA.The X-axis of FIG. 3 is the fluorescence intensity, minimum is zero andmaximum is 4095. The Y-axis of FIG. 3 is the normalized distribution(percentage) of the cell population.

FIG. 4 shows two examples of the stable cell lines established bytransfection with hPtFP green fluorescent protein DNA of the presentinvention. The left panel shows stable A549 cells transfectantsexpressing hPtFP. The right panel shows stable HEK293 cell transfectantsexpressing hPtFP.

FIG. 5 shows the non toxic effect of hPtFP on its mammalian host cells.

FIG. 6, Panel A, shows COS1 cells transiently transfected with human CD7and were stained with a monoclonal antibody against human CD7. Panels B& C show COS1 cells transiently transfected with CD7-fluorescentprotein.

FIGS. 7 and 8 show a diagram of the configuration of the Caspase 3 andCaspase 8 biosensors, respectively.

FIG. 9 shows the wild type Ptilosarcus gurneyi nucleotide sequence (toprow) compared to the nucleotide sequence encoding the humanizedPtilosarcus gurneyi fluorescent protein of the present invention (bottomrow).

FIG. 10 shows the amino acid sequence (top row) and the double strandednucleotide sequence (bottom rows) of the humanized Ptilosarcus gurneyifluorescent protein of the present invention.

FIG. 11 shows the full length nucleotide of humanized Ptilosarcusgurneyi fluorescent protein of the present invention including regionsupstream and downstream to the coding region.

FIG. 12 shows the full length protein sequence of humanized Ptilosarcusgurneyi fluorescent protein of the present invention from start codon tostop codon.

FIG. 13 shows the (truncated) deletion mutants of hPtFP and theireffects on green fluorescence.

FIG. 14 shows HeLa cells transfected with the hPtFP-Caspase-8 biosensorof this invention before and after treatment with staurosporine.

FIG. 15 shows a codon usage table for a human system, compiled from22747 coding regions CDS's (10965560 codons).

FIG. 16 shows a restriction endonuclease cleavage map for expressionvector M2.

FIG. 17 shows the nucleotide sequence of expression vector M2.

FIG. 18 shows a general description of gene synthesis.

SUMMARY OF THE INVENTION

The present invention has met the hereinbefore described needs. Thepresent invention provides an isolated and purified DNA encoding a greenfluorescent protein from the orange seapen Ptilosarcus gurneyi in whichall of the codons are favored for mammalian systems. The full lengthencoded protein of the present invention has 239 amino acid residues.Preferably, the encoded protein of the present invention is truncatedhaving 224 amino acid residues and most preferably has 219 amino acidresidues. In comparison to the wild type Ptilosarcus gurneyi greenfluorescent protein having 238 amino acid residues, codons for 145 aminoacids of the humanized Ptilosarcus gurneyi green fluorescent protein ofthe present invention were changed based on human codon bias.

The encoded protein of the present invention, when expressed inmammalian cell lines gives strong green fluorescence.

The synthetic DNA of the present invention can be used as a fluorescenttag for monitoring the activities of fusion partners using known imagebased techniques.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a synthetic cDNA, based on the orangeseapen Ptilosarcus gurneyi green fluorescent protein sequence, to encodea green fluorescent protein in which the majority of the codons are thefavored or the most favored codons for mammalian expression systems.This process of codon preference modification when going from a nativespecies to a host species, especially of going into a human cell-linehost, is called “humanization”.

A humanized gene is one that has been adapted for expression in humancells by replacing at least one, and most preferably, a significantnumber of the codons in the native gene codons with codons that are mostfrequently used in human gene expression. Thus, the native codon usageis replaced with a codon that is more favorable for translation in ahuman or mammalian cell line. One reason for low expression of foreigngenes in mammalian expression systems is the poor translation efficiencyof the mRNA in the mammalian, and especially human cell environment. Thereason for this is the difference in abundance of particular isoacceptortRNA's that are different in human cells than those found in otherorganisms. In this instance the isoacceptor tRNA's are different in thePtilosarcus gurneyi orange seapen than in human cells. Making the codonusage in the foreign gene match the prevalent isoacceptor tRNAsubpopulation leads to improved translation efficiency, thus improvedexpression of the foreign gene in human cells.

The use of codon preference modification at the cDNA level results inhigher levels of expression of the modified DNA molecule. Higher levelsof expression leads to higher protein yield thus higher fluorescentsignal in mammalian cells expressing the modified cDNA. The encodedprotein of the present invention has 239 amino acid residues. Incomparison to the wild type Ptilosarcus gurneyi green fluorescentprotein (238 amino acid residues), codons for 145 amino acids werechanged based on human codon preferences. One amino acid (valine) wasadded at the amino terminus to be the second amino acid residue in thisprotein. The encoded protein, when expressed in mammalian cell lines,gives strong green fluorescence. Generally, the green fluorescence to beachieved by the present invention is the production of light visible tothe naked eye for qualitative purposes. Thus, the amount of thecomponent of the bioluminescence reaction need not be stringentlydetermined or met. It must be sufficient to produce light. The syntheticDNA of the present invention can be used as a fluorescent tag formonitoring the activities of its fusion partner, as described herein,using known imaging based approaches.

As will be appreciated by those skilled in the art, gene synthesis isperformed by piecing together small pieces of double-stranded syntheticDNA. Each small double-stranded synthetic DNA is pieced together witheven smaller single-stranded oligonucleotides. To place together onepiece of the double-stranded DNA, 4 oligonucleotides are required. FIG.18 shows oligo 1 and oligo 2 are complimentary to each other in part ofthem. Thus, they can anneal to each other and can be extended by a DNApolymerase, such as for example Taq polymerase. The extended templatethen works as the template for an amplification reaction (using, forexample. Taq polymerase) employing oligo 3 and oligo 4 as the sense andanti-sense primers, respectively. The extension of the template and theamplification reaction are actually performed at the same time in thesame test tube, and thus no separate step is shown in FIG. 18. Twopieces of double-stranded synthetic DNA, from a synthesis scheme as forexample the synthesis scheme set forth in FIG. 18, may serve as thetemplate for a new round of synthesis as long as they contain anoverlapping region that can anneal to each other. This process may berepeated several times in order to create a long synthetic gene that cannot be synthesized in one step. See J Hass et al., Codon usagelimitation in the expression of HIV-1 envelope glycoprotein, Curr.Biol., Vol 6 (3), pages 315–324 (March 1996).

The fluorescent protein encoded by the modified humanized cDNA of thepresent invention is substantially identical to the wild typePtilosarcus gurneyi fluorescent protein at the amino acid level, withthe exception that the present invention provides for the addition of asingle valine residue at position number 2 from the amino terminus. Theabsorption and emission spectra of the hPtFP in COS-1 cells wasunchanged as compared to the wildtype Ptilosarcus gurneyi.

FIG. 10 shows the double stranded nucleotide sequence (bottom two rows)of the entire coding region and the deduced amino acid sequence (toprow) of the humanized Ptilosarcus gurneyi fluorescent protein(hereinafter “hPtFP”) of the present invention, as well as the start andstop codon sequence, untranslated regions and restriction sites. FIG. 11shows the full length coding sequence of hPtFP of the present inventionincluding regions upstream and downstream to the coding region.

FIG. 12 shows the full length coding sequence of the hPtFP of thepresent invention from start codon to stop codon. Thus the total lengthof the humanized Ptilosarcus gurneyi fluorescent protein (hPtFP) of thepresent invention is 239 amino acid residues versus 238 for the wildtype Ptilosarcus gurneyi fluorescent protein (PtFP). It will beappreciated that at the nucleotide level, approximately 61% of the wildtype codons have been changed based on human codon bias. FIG. 15 showsthe codon usage table for human system, compiled from 22747 CDS's(10965560 codons) based on GenBank Release 118.0 (Jun. 15, 2000),obtained from Kazusa DNA Research Institute (Japan). FIG. 15 shows thefollowing fields: [triplet] [amino acid] [fraction, % of gene using theparticular codon] [number of codons examined].

FIG. 9 shows the wild type Ptilosarcus gurneyi (PtFP) nucleotidesequence (top row) compared to the nucleotide sequence (bottom row)encoding the hPtFP of the present invention. The difference at thenucleotide level (versus codon level) is that the hPtFP open readingframe (including the stop codon) contains 720 nucleotides, whereas thePtFP open reading frame (including the stop codon) contains 717nucleotides. It will be appreciated by those skilled in the art, thatwithout counting the extra valine introduced into hPtFP of the presentinvention, there are 166 nucleotide differences between the 717nucleotides compared, or 23.15 percent difference (or 76.85% identity).If the stop codon is excluded in this comparison, there are 165nucleotide differences in the 714 nucleotides compared, or 23.10 percentdifference (or 76.9% identity). The synthetic DNA of the presentinvention was subcloned into an expression vector M2 (Cellomics, Inc.,Pittsburgh, Pa., USA) after restriction digestion of both DNAs withHindIII (New England Biolabs, Inc., Beverly, Mass., USA) and NotI (NewEngland Biolabs, Inc., Beverly, Mass., USA) restriction endonucleases.The resulting expression vector was then used to transfect COS1 cells(CRL-1650, American Type Culture Collection [ATCC], Manassas, Va., USA)using FUGENE 6 transfection reagent (Roche Molecular Biochemicals,Indianapolis, Ind., USA) following the protocol supplied by themanufacturer. FIG. 16 shows an restriction endonuclease cleavage map ofM2. FIG. 17 shows the coding sequence of M2. M2 is a derivative ofpCI-neo (Promega, Madison, Wis., USA). Forty-eight (48) hours posttransfection, the fluorescence was observed with an invertedepi-fluorescent microscope using a filter set for observing fluorescenceas set forth in FIG. 1. FIG. 1 shows the expression of hPtFP in COS1cells transiently transfected with the synthetic DNA of the presentinvention (right panel). The cells were counter stained with Hoechst33342 (Molecular Probes, Eugene, Oreg., USA), a nuclear stain, FIG. 1(left panel).

Forty-eight hours after initial transfection with the synthetic greenfluorescent protein DNA of the present invention, COS1 cells weretrypsinized and were kept in suspension. The absorption and emissionspectra of the live cells expressing the hPtFP of the present inventionwere then measured, as shown in FIG. 2. FIG. 2 shows the in situfluorescence of the humanized Ptilosarcus gurneyi fluorescent protein(hPtFP) in COS1 cells.

To compare the expression of wild type Ptilosarcus gurneyi fluorescentprotein DNA with humanized Ptilosarcus gurneyi green fluorescent proteinsynthetic DNA as described above, an expression vector for the wild typePtilosarcus gurneyi DNA was constructed by cloning the wild type PtFPinto the expression vector M2, and thus, these two expression vectorsunder comparison differed only in their coding regions. Both DNAconstructs were purified using QIAGEN plasmid kit (QIAGEN Inc.,Valencia, Calif., USA) folowing the instructions supplied by themanufacturer. The purified DNA preps were quantitated by reading theoptic absorption at 260 nm (nanometers) with a HP8453 UV—visiblespectrophotometer (Agilent Technologies, Palo Alto, Calif., USA) andcalculated based on 1 O.D.=50 ng (nanograms) DNA. An identical amount ofwild type Ptilosarcus gurneyi fluorescent protein DNA and the hPtFP wasused to transfect COS1 cells, respectively, under identical conditionsusing FUGENE6 reagent, as described above. Forty hours post-transfectioncells were fixed with 3.7 percent formaldehyde in the presence of 10micrograms/milliliter of Hoechst 33342 (Molecular Probes, Eugene, Oreg.,USA). The fluorescent images of the cells were then acquired usingARRAYSCAN II instrument (Cellomics, Inc., Pittsburgh, Pa., USA) with 10×objective and filter setting at “FITC broad” (excitation=365+/−25 nm,emission=450+/−30 nm for Hoechst 33342—for fluorescent stain of nuclei,and excitation=475+/−20 nm, emission=535+/−22.5 nm for hPtFP). U.S. Pat.No. 5,989,835 describes the ARRAYSCAN II optical system and isincorporated by reference herein. The acquired images were then analyzedusing a desktop client of ARRAYSCAN II instrument by identifying thenuclear region, and the intensity of the hPtFP was measured in theidentified nuclear area. FIG. 3 shows the comparative fluorescencemeasurements of COS1 cells transfected with the humanized cDNA of thepresent invention and the wild type cDNA. FIG. 3 shows that thesynthetic Ptilosarcus gurneyi green fluorescent protein DNA of thepresent invention emits stronger fluorescent signals than the wild typePtilosarcus gurneyi green fluorescent protein. The results shown in FIG.3 confirm the Applicant's visual observation (qualitative) that thehPtFP DNA of the present invention produces more hPtFP expressing cellsand brighter cells than the wild type “PtFP” DNA in transienttransfection.

The humanized synthetic DNA does not have toxic effects on the hostcells, which aids in its increased stable expression. Stable expressionof the hPtFP of the present invention was achieved in HEK293 (CRL-1573ATCC, Manassas, Va., USA) and A549 (CCL-185, ATCC, Manassas, Va., USA)cell lines. The cells were co-transfected with the hPtFP construct andpSV2-neo (Cat #37149, ATCC, Manassas, Va., USA) with FUGENE 6transfection reagent (Roche Molecular Biochemicals, Indianapolis, Ind.,USA) following the manufacturer's instructions. Two days aftertransfection, cells were treated with 0.4 mg/ml (milligram/milliliter)G418 in normal growth medium. After treating with G418 for two weeks,drug resistant cells were isolated or pooled. A mixture of stablytransfected HEK293 mixed population (in which about 30% of the cellsexpressing hPtFP of this invention) were plated out in 96 cell microplates. After 2, 4, or 6 days incubation, the cells were fixed with 3.7%formaldehyde for 20 minutes at room temperature (25° Centigrade). Thepercentage of positive cells was quantitated with ARRAYSCAN IIinstrument, as described herein.

FIG. 4, left panel, shows stably transfected HEK293 cells expressing thehPtFP of the present invention, and the right panel shows stablytransfected A-549 cells. To compare the growth rates of cells expressingor not expressing hPtFP, a mixture of stably transfected HEK293 mixedpopulation (in which about 30% of the cells expressing hPtFP of thisinvention) were plated out in 96-well micro plates. After 2, 4, or 6days incubation, the cells were fixed with 3.7% (percent) formaldehydefor twenty minutes at room temperature (25 degrees Centigrade). Thepercentage of positive cells was quantitated with ARRAYSCAN IIinstrument, as described herein. FIG. 5 shows the quantitation ofpositive cells from a mixture of stably transfected HEK293 cellpopulation wherein approximately thirty percent (30%) of the cellsexpressing hPtFP were plated and incubated as described above. FIG. 5shows that the percentage of positive cells did not change during cellpassage under no selection, indicating that the expression of thehumanized Ptilosarcus gurneyi fluorescent protein of this invention isnot toxic to the cells.

The hPtFP of this invention is useful as a fusion partner for taggingpurposes. The hPtFP of this invention was fused to a model type-I singlespan transmembrane protein, human CD7 linked to the reactant targetdomain of the C-terminus of CD7. Human CD7 is a type-I single spantransmembrane protein. CD7 is a member of the immunoglobulin genesuperfamily well known by those skilled in the art and is a reliableclinical marker of T-cell acute lymphocytic leukemia. The fusion proteinwas expressed in COS1 cells by transient transfection using FUGENE 6reagent, described hereinbefore, following the protocol supplied by themanufacturer. The distribution of the chimeric protein is similar to CD7(no fusion partner) when transiently expressed by COS1 cells.

FIG. 6, Panel A, shows C0S1 cells transiently transfected with human CD7and stained with a monoclonal antibody against human CD7 (CD7 Ab-2,clone 124-1D1, Labvision Corp., Freemont, Calif., USA). FIG. 6, Panels Band C show COS1 cells transiently transfected with CD7-hPtFP of thisinvention. Panel B shows staining using a monoclonal antibody againsthuman CD7 and Panel C shows a direct observation of the CD7-hPtFP fusionprotein. The CD7-hPtFP chimera exhibits comparable localization as theuntagged CD7.

The hPtFP is also useful in constructing biosensor systems. For example,the hPtFP may be used to construct protease biosensors for which thebasic principle of the protease biosensors is to spatially separate thereactants from the products generated during a proteolytic reaction. Theseparation of products from reactants occurs upon proteolytic cleavageof the protease recognition site within the biosensor, allowing theproducts to bind to, diffuse into, or be imported into compartments ofthe cell different from those of the reactant. This spatial separationprovides a means of quantitating a proteolytic process directly inliving or fixed cells. A design of the biosensor provides a means ofrestricting the reactant (uncleaved biosensor) to a particularcompartment by a protein sequence (“reactant target sequence”) thatbinds to or imports the biosensor into a compartment of the cell. Thesecompartments include, but are not limited to any cellular substructure,macromolecular cellular component, membrane-limited organelles, or theextra-cellular space. Given that the characteristics of the proteolyticreaction are related to product concentration divided by the reactantconcentration, the spatial separation of products and reactants providesa means of uniquely quantitating products and reactants in single cells,allowing a more direct measure of proteolytic activity.

The molecular based biosensors may be introduced into cells viatransfection and the expressed chimeric proteins analyzed in transientlytransfected cell populations or stable cell lines. They may also bepre-formed, for example by production in a prokaryotic or eukaryoticexpression system, and the purified protein introduced into the cell viaa number of physical mechanisms including, such as for example, but notlimited to, micro-injection, scrape loading, electroporation, andsignal-sequence mediated loading, etc.

Measurement modes may include, such as for example, but are not limitedto, the ratio or difference in fluorescence, luminescence, orphosphorescence: (a) intensity; (b) polarization; or (c) lifetime,between reactant and product. These latter modes require appropriatespectroscopic differences between products and reactants. For example,cleaving a reactant containing a limited-mobile signal into a very smalltranslocating component and a relatively large non-translocatingcomponent may be detected by polarization. Alternatively, significantlydifferent emission lifetimes between reactants and products allowdetection in imaging and non-imaging modes.

One example of a family of enzymes for which this biosensor can beconstructed to report activity is the caspase family. Caspases are aclass of proteins that catalyze proteolytic cleavage of a wide varietyof targets during apoptosis. Following initiation of apoptosis, theClass II “downstream” caspases are activated and are the point of noreturn in the pathway leading to cell death, resulting in cleavage ofdownstream target proteins. Specifically, the biosensors describedherein are engineered to use nuclear translocation of cleaved hPtFP as ameasurable indicator of caspase activation. Additionally, the use ofspecific recognition sequences that incorporate surrounding amino acidsinvolved in secondary structure formation in naturally occurringproteins may increase the specificity and sensitivity of this class ofbiosensor.

The protein biosensors herein disclosed can be adapted to report theactivity of any member of the caspase family of proteases, as well asany other protease, by a substitution of the appropriate proteaserecognition site in any of the constructs. These biosensors can be usedto detect in vivo activation of enzymatic activity and to identifyspecific activity based on cleavage of a known recognition motif. Thisscreen can be used for both live cell and fixed end-point assays, andcan be combined with additional measurements to provide amulti-parameter assay, as is well known in the art.

Thus, another aspect of the present invention provides recombinantnucleic acids encoding a protease biosensor, comprising: (a) a firstnucleic acid sequence encoding a Ptilosarcus gurneyi green fluorescentprotein having its codon usage optimized for expression in human cellsthat encodes at least one detectable polypeptide signal; (b) a secondnucleic acid sequence that encodes at least one protease recognitionsite, wherein the second nucleic acid sequence is operatively linked tothe first nucleic acid sequence that encodes at least one detectablepolypeptide signal; and (c) a third nucleic acid sequence that encodesat least one reactant target sequence, wherein the third nucleic acidsequence is operatively linked to the second nucleic acid sequence thatencodes at least one protease recognition site.

Generally, a protease biosensor is composed of multiple domains,including at least a first detectable polypeptide signal domain, atleast one reactant target domain, and at least one protease recognitiondomain, wherein the detectable signal domain and the reactant targetdomain are separated by the protease recognition domain. Thus, the exactorder is not generally critical as long as the protease recognitiondomain separates the reactant target and first detectable signal domain.For each domain, one or more of the specified sequences is present.

The organizations of the biosensors are shown in FIG. 7 (Caspase 3) andFIG. 8 (Caspase 8). Those persons skilled in the art will recognize thatany one of a wide variety of protease recognition sites, reactant targetsequences, polypeptide signals, and/or product target sequences can beused in various combinations in the protein biosensor of the presentinvention, by substituting the appropriate coding sequences into themulti-domain construct. Non-limiting examples of such alternativesequences are shown in FIGS. 7 and 8. Similarly, those skilled in theart will recognize that modifications, substitutions, and deletions canbe made to the coding sequences and the amino acid sequences of eachindividual domain within the biosensor, while retaining the function ofthe domain. Such various combinations of domains and modifications,substitutions and deletions to individual domains are within the scopeof the instant invention.

As used herein, the term “coding sequence” or a sequence which “encodes”a particular polypeptide sequence, refers to a nucleic acid sequencewhich is transcribed (in the case of DNA) and translated (in the case ofmRNA) into a polypetide in vitro or in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Acoding sequence can include, such as for example, but is not limited to,cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

As used herein, the term DNA “control sequences” refers collectively topromoter sequences, ribosome binding sites, polyadenylation signals,transcription termination sequences, upstream regulatory domains,enhancers, and the like, which collectively provide for thetranscription and translation of a coding sequence in a host cell. Notall of these control sequences need always be present in a recombinantvector so long as the DNA sequence of interest is capable of beingtranscribed and translated appropriately.

As used herein, the term “operatively linked” refers to an arrangementof elements wherein the components so described are configured so as toperform their usual function. Thus, control sequences operatively linkedto a coding sequence are capable of effecting the expression of thecoding sequence. The control sequences need not be contiguous with thecoding sequence, so long as they function to direct expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operatively linked” tothe coding sequence.

Furthermore, a nucleic acid coding sequence is operatively linked toanother nucleic acid coding sequence when the coding region for bothnucleic acid molecules are capable of expression in the same readingframe. The nucleic acid sequences need not be contiguous, so long asthey are capable of expression in the same reading frame. Thus, forexample, intervening coding sequences, and the specified nucleic acidcoding regions can still be considered “operatively linked”.

The intervening coding sequences between the various domains of thebiosensors can be of any length so long as the function of each domainis retained. Generally, this requires that the two dimensional andthree-dimensional structure of the intervening protein sequence does notpreclude the binding or interaction requirements of the domains of thebiosensor, such as product or reactant targeting, binding of theprotease of interest to the biosensor, fluorescence or luminescence ofthe detectable polypeptide signal, or binding of fluorescently labeledepitope-specific antibodies.

Within this application, unless otherwise noted, the techniques utilizedmay be found in any of several well-known references such as MolecularCloning: A Laboratory Manual (Sambrook, et al. 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185 edited by D. Goeddel, 1991, Academic Press, San Diego, Calif.),“Guide to Protein Purification” in Methods in Enzymology (M. P.Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of animal Cells: A Manual of Basic Technique, 2nd Ed.(R. I. Freshney, 1987. Liss, Inc. New York, N.Y.) Gene Transfer andExpression Protocols, pp. 109–128, ed. E. J. Murray, The Human PressInc. Clifton, N.J.), and the Ambion Catalog (Ambion, Austin, Tex.).

The biosensors of the present invention are constructed and used totransfect host cells using standard techniques in the molecularbiological arts. Any number of such techniques, all of which are withinthe scope of this invention, can be used to generate proteasebiosensor-encoding DNA constructs and genetically transfected host cellsexpressing the biosensors. The biosensors disclosed in pending publishedpatent application WO 0026408, entitled “A System For Cell BasedScreening” provide examples of such biosensors; PCT/US99/25431 is madeof record and incorporated by reference into this patent application.The non-limiting examples that follow demonstrate one such technique forconstructing the biosensors of the invention. For example, by changingthe protease recognition sequence of the sensors shown herein into therecognition sequence for other caspases or other intracellularproteases, such as for example calpain and cathepsins, new specificprotease sensors can easily be generated. Other examples of greenfluorescent protein-based biosensors include, but are not limited to,fluorescent resonance energy transfer (FRET) based, green fluorescentprotein-based caspase sensor disclosed by J. Jones et al., J. Biomol.Screen., Vol. 5 (5), pages 307–318 (October 2000), A. Miyawaki et al.,Nature, Vol. 388 (6645), pages 882–887 (August 1997), and J. P. Waud etal., J. Biochem., Vol. 357 (Pt. 3), pages 687–697 (August 2001).

In addition to the full length coding sequence hPtFP of the presentinvention as shown in Seq. ID No. 1 several truncation mutants aredisclosed ranging from truncations at the 5′ (amino) terminus totruncations at the 3′ (carboxy) terminus. SEQ ID No. 2 shows the aminoacid sequence of the full length hPtFP of the present invention. SEQ IDNo. 3 shows a truncation mutant of the present invention wherein thetruncation occurs at the 3′ (carboxy) terminus, specifically, includingamino acid sequence 1–224. SEQ ID No. 4 shows a truncation mutant of thepresent invention wherein the truncation occurs at the 5′ (amino)terminus, specifically including amino acid sequence 10–229. FIG. 13shows deletion mutants of the hPtFP of the present invention that wereconstructed. The fluorescent intensity upon visual inspection of eachconstruct is shown in FIG. 13. The deletion mutants of the hPtFP of thepresent invention were constructed and transiently transfected into HeLacells. The deletion mutants were created by employing PCR (polymerasechain reaction) technology, as known by those skilled in the art, andwere sub-cloned into the expression vector M2 in which the expression inthe mammalian systems is driven by the CMV (cytomegalovirus) promoter.All mutants and the full length hPtFP of this invention, expressionconstructs were designed to be identical in the non-coding region. Allcoding regions constructed for this comparison as shown in FIG. 13retain M (methionine) and V (valine) as the first and second aminoacids, respectively. The plasmids were then transfected into Hela cellsand observed for fluorescence 24 hours after transfection as shown inFIG. 13. It will be appreciated by those skilled in the art that thetruncation mutants of the present invention may be employed asfluorescent tags for monitoring the activities of its fusion partnersusing an image based approach as a biosensor.

FIG. 14 shows HeLa cells (CCL-2, ATCC, 10801, Manassas, Va., USA)transfected with with the hPtFP-Caspase-8 biosensor with FUGENE 6reagent (Roche Molecular Biochemicals, Indianapolis, Ind., USA). Twentyfour hours after transfection, the HeLa cells were treated withstaurosporine (Sigma-Aldrich, St. Louis, Mo., USA), at 1 nM (nano molar)or 10 nM. Fluorescent signals from the cells were observed at the 6hours and 24 hours, respectively, after addition of the staurosporine tothe medium.

Whereas particular embodiments of this invention have been describedherein for purposes of illustration, it will be evident to those personsskilled in the art that numerous variations of the details of thepresent invention may be made without departing from the invention asdefined in the appended claims that follow the SEQUENCE LISTING.

1. An isolated and purified green fluorescent protein truncated mutantfrom Ptilosarcus gurneyi having at least one amino acid deletionoccurring within amino acids 3–10 of SEQ ID NO: 2 wherein the amino acidat position number 1 is methionine and at position number 2 is valine.2. The isolated and purified green fluorescent protein truncated mutantof claim 1 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 5. 3. The isolated and purified green fluorescent proteintruncated mutant of claim 1 wherein the amino acid sequence is the aminoacid sequence of SEQ ID NO:
 6. 4. The isolated and purified greenfluorescent protein truncated mutant of claim 1 wherein the amino acidsequence is the amino acid sequence of SEQ ID NO:
 7. 5. The isolated andpurified green fluorescent protein truncated mutant of claim 1 whereinthe amino acid sequence is the amino acid sequence of SEQ ID NO:
 8. 6.The isolated and purified green fluorescent protein truncated mutant ofclaim 1 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 9. 7. The isolated and purified green fluorescent proteintruncated mutant of claim 1 wherein the amino acid sequence is the aminoacid sequence of SEQ ID NO:
 10. 8. The isolated and purified greenfluorescent protein truncated mutant of claim 1 wherein the amino acidsequence is the amino acid sequence of SEQ ID NO:
 11. 9. The isolatedand purified green fluorescent protein truncated mutant of claim 1wherein the amino acid sequence is the amino acid sequence of SEQ ID NO:12.
 10. An isolated and purified green fluorescent protein fromPtilosarcus gurneyi having the truncated coding sequence of SEQ ID NO:3.
 11. An isolated and purified green fluorescent protein fromPtilosarcus gurneyi having the truncated coding sequence of SEQ ID NO:4.
 12. The isolated and purified green fluorescent protein truncatedmutant from Ptilosarcus gurneyi having at least one amino acid deletionoccurring within amino acids 225–239 of SEQ ID NO: 2 wherein the aminoacid at position 1 is methionine and at position number 2 is valine. 13.The isolated and purified green fluorescent protein truncated mutant ofclaim 12 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 13. 14. The isolated and purified green fluorescent proteintruncated mutant of claim 12 wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:
 14. 15. The isolated and purifiedgreen fluorescent protein truncated mutant of claim 12 wherein the aminoacid sequence is the amino acid sequence of SEQ ID NO:
 15. 16. Theisolated and purified green fluorescent protein truncated mutant ofclaim 12 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 16. 17. The isolated and purified green fluorescent proteintruncated mutant of claim 12 wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:
 17. 18. The isolated and purifiedgreen fluorescent protein truncated mutant of claim 12 wherein the aminoacid sequence is the amino acid sequence of SEQ ID NO:
 18. 19. Theisolated and purified green fluorescent protein truncated mutant ofclaim 12 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 19. 20. The isolated and purified green fluorescent proteintruncated mutant of claim 12 wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:
 20. 21. The isolated and purifiedgreen fluorescent protein truncated mutant of claim 12 wherein the aminoacid sequence is the amino acid sequence of SEQ ID NO:
 21. 22. Theisolated and purified green fluorescent protein truncated mutant ofclaim 12 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 22. 23. The isolated and purified green fluorescent proteintruncated mutant of claim 12 wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:
 23. 24. The isolated and purifiedgreen fluorescent protein truncated mutant of claim 12 wherein the aminoacid sequence is the amino acid sequence of SEQ ID NO:
 24. 25. Theisolated and purified green fluorescent protein truncated mutant ofclaim 12 wherein the amino acid sequence is the amino acid sequence ofSEQ ID NO:
 25. 26. The isolated and purified green fluorescent proteintruncated mutant of claim 12 wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:
 26. 27. The isolated and purifiedgreen fluorescent protein truncated mutant of claim 12 wherein the aminoacid sequence is the amino acid sequence of SEQ ID NO:
 27. 28. Theisolated and purified green fluorescent protein truncated mutant fromPtilosarcus gurneyi having at least one amino acid deletion occurringwithin amino acids 3–10 and at least one amino acid deletion occurringwithin amino acids 225–239 of SEQ ID NO: 2 wherein the amino acid atposition number 1 is methionine and at position number 2 is valine.